The present invention is in the field of recombinant genetics. In particular, the present invention relates to a novel receptor ligand, designated Fhm, belonging to the TNF ligand supergene family and nucleic acid molecules encoding the same. The invention also relates to vectors, host cells, anti-Fhm antibodies, and recombinant methods for producing Fhm polypeptides. The invention also relates to the use of the recombinant Fhm polypeptide to identify putative binding partners/receptors. In addition, provided for are methods and reagents for the diagnosis of diseases associated with abnormal Fhm or abnormal expression of its putative receptor, and methods and pharmaceutical compositions for the treatment of diseases associated with abnormal Fhm or abnormal expression of Fhm and/or itsreceptor. The invention also discloses pharmaceutical compositions for use in the treatment of these diseases.
Technical advances in the identification, cloning, expression and manipulation of nucleic acid molecules have greatly accelerated the discovery of novel therapeutics based upon deciphering the human genome. Rapid nucleic acid sequencing techniques can now generate sequence information at unprecedented rates, and coupled with computational analyses, allow the assembly of overlapping sequences into entire genome and the identification of polypeptide-encoding regions. Comparison of a predicted amino acid sequence against a database compilation of known amino acid sequences can allow one to determine the extent of homology to previously identified sequence and/or structure landmarks. Cloning and expression of a polypeptide-encoding region of a nucleic acid molecule provides a polypeptide product for structural and functional analysis. Manipulation of nucleic acid molecules and encoded polypeptides to produce variants and derivatives thereof may confer advantageous properties on a product for use as a therapeutic.
However, in spite of the significant technical advances in genome research over the past decade, the potential for development of novel therapeutics based on the human genome is still largely unrealized. While a number of genes encoding potentially beneficial protein therapeutics, or those encoding polypeptides which may act as xe2x80x9ctargetsxe2x80x9d for therapeutic molecules, have been identified using recombinant DNA technology, the structure and function of a vast number of genes in the genome of mammals are yet unknown.
Identification and Characterization of TNF-Family of Ligands and Receptors
Tumor necrosis factor (TNF) was first identified in the serum of mice and rabbits which had been infected with bacillus of Calmette and Guerin(BCG) and which had been injected with endotoxin. TNF activity in the serum of these animals was recognized on the basis of its cytotoxic and anti-tumor activities. This TNF activity, referred to as TNF-xcex1, is produced particularly by activated monocytes and macrophages, and has been implicated in normal growth processes as well as in a variety of diseases.
Following the discovery of TNF-xcex1, independent research led to the identification of another cytokine associated with inflammatory responses lymphotoxin-xcex1 (LT-xcex1), which was shown to be produced exclusively by lymphocytes. LT-xcex1 was subsequently shown to be 30% homologous with TNF-xcex1, and was renamed TNF-xcex2. It is now clear that TNF-xcex1 and TNF-xcex2 are members of a gene family that includes yet another member termed LT-xcex2 (Browning et al., Cell 72:847-856, 1993). The three genes are tightly linked within the MHC complex and show similar organization. Moreover, the biologically active forms of TNF-xcex1 and TNF-xcex2 are homotrimers and share many of the same biological activities including competing for the same cell-surface receptors (Agarwal et al., Nature 318:665-667, 1985). Two distinct but structurally homologous receptors have been identified, and each has been shown to bind both the ligands and mediate their effects.
However, it has been recognized that TNFs are only representative members of the rapidly expanding supergene familiy that includes TNF-xcex1, TNF-xcex2/lymphotoxin-xcex1 (LT-xcex1), lymphotoxin-xcex2 (LT-xcex2), FasL, CD40L, CD30L, CD27L, 4-1BBL, and TNF-related apoptosis-inducing ligand (TRAIL), RANKL, GITRL and TNF-2. See generally Orlinick et al., Cell Signal, 10(8):543-551 (1998). The distinctive but overlapping cellular responses induced by members of the TNF family of ligands following their interaction(s) with their cognate cell-surface receptors result in clearly defined developmental and regulatory changes in cells of the lymphoid, hematopoietic, and other lineages. For example, the TNF family of ligands are involved in growth regulation and differentiation of cells which are involved in inflamation, immune processes and hematopoiesis (Bayert, R. and Fiers, W., Tumor Necrosis Factor and Lymphokines in: Cytokines eds. Anthony Mire-Sluis and Robin Thorpe, Academic Press San Diego Calif., 1998). The TNF family of ligands activates the immune defenses against parasites, and act directly or indirectly as mediators in immune reactions and inflammatory processes. However, administration of TNF and/or other members of the TNF family can also be accompanied by harmful phenomena such as shock and tissue damage (Bayert, R. and Fiers, W., supra). The main physiological role of the TNF family of ligands is the activation of first-line reaction of an organism to microbial, parasitic, viral infections, or to mechanical stress and cancer. For example, TNF-related apoptosis-inducing ligand (TRAIL) has been demonstrated to induce apoptosis of a number of different types of cancer cells as well as virally infected cells.
Furthermore, a number of observations have also led to the conclusion that the TNF family of ligands are also involved in a variety of pathological conditions including cachexia, toxic shock syndrome, inflammatory diseases such as rheumatoid and osteoarthritis, and in lethality resulting from graft-versus-host reaction (GVHR) rapid necrosis of tumors, apoptosis, immunostimulation and resistance to parasites and viruses. (Bayert, R. and Fiers, W., supra).
Like other cytokines, the members of the TNF family of ligands act via specific cell-surface receptors. The receptors, with two exceptions, are type 1 membrane-associated proteins. Sequence homology amongst them is almost entirely confined to their extracellular domains. For example, two TNF receptors have been cloned which differ in size and in binding affinity (Bayert, R. and Fiers, W., supra). Both receptors bind TNF-xcex1 and TNF-xcex2 and are membrane associated proteins. The two receptors consist of extracellular domains which bind TNF (and are homologous for 28%), single transmembrane domains, and intracellular domains which are totally different from each other and which do not contain any recognizable structural motifs that have been associated with any particular function. Based on similarities in their extracellular domains, these receptors belong to a receptor gene superfamily that includes the low-affinity nerve growth factor (NGF) receptor, the Fas antigen, the TNFR-RP, TRAIL-R, PV-A53R, RANK, GITR, and the OX40 antigen found on activated T-cells (Smith et al., Cell, 76(6):959-962, 1994; Baker and Reddy, Oncogene, 12(1):1-9, 1996).
In addition to the membrane associated receptor molecules described above, a number the receptors belonging to the TNF-receptor supergene family exist as soluble ligand binding proteins. Many of the soluble forms of the transmembrane receptors were subsequently identified as containing only the extracellular ligand binding domain(s) of the receptors. For example, a soluble form of TNF receptor has been found in urine and serum (See U.S. Pat. No.: 5,843,789 and Nophar et al., EMBOJ., 9(10):3269-3278, 1990), and has been shown to arise by proteolytic cleavage of cell surface TNF-receptors (Wallach et al., Agents Actions Suppl., 35:51-57 1991). These soluble forms of receptor molecules have been implicated in the modulation of TNF activity by not only interfering with TNF binding to its receptor, but also by stabilizing the TNF structure and preserving its activity, thus prolonging some of its effects (Aderka et al, Cytokine and Growth Factor Reviews, 7(3):231-240, 1996).
The activity of members of the TNF family of ligands is tightly regulated at the levels of secretion and receptor expression. Additional regulatory mechanisms are provided by action of specific inhibitory proteins present on cell surfaces and in biological fluids. While some of these inhibitory proteins have been identified as soluble forms of receptor molecules, the identity of many of these cytokine regulatory proteins are as yet unknown. However, abnormalities in the production of these substances might contribute to the pathophysiology of a variety of diseases including immune and neoplastic diseases. Besides their role in regulating cytokine activity in vivo, these regulatory molecules hold significant potential for therapeutic use as very specific inhibitors/anti-cytokine agents, and as indicators in diagnosis and assessment of immune function and growth parameters in a variety of autoimmune and malignant diseases.
Because of the important role of the TNF family of ligands (and their receptors) in health and disease, a need exists to identify, isolate, and characterize additional members of the family, for use in diagnosing and treating disease and pathological conditions.
The present invention relates to a novel serine/threonine kinase family and uses thereof. More specifically, the present invention relates to novel Fhm nucleic acid molecules and encoded polypeptides, and uses thereof.
The invention provides for an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO: 3;
(b) a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 4;
(c) a nucleotide sequence which hybridizes under moderately or highly stringent conditions to the complement of (a) or (b), wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 3; and
(d) a nucleotide sequence complementary to any of (a) through (c).
The invention also provides for an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence encoding a polypeptide that is at least about 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 percent identical to the polypeptide set forth in 4, wherein the polypeptide has an activity of the encoded polypeptide set forth in SEQ ID NO: 4 as determined using a computer program selected from the group consisting of GAP, BLASTP, BLASTN, FASTA, BLASTA, BLASTX, BestFit, and the Smith-Waterman algorithm;
(b) a nucleotide sequence encoding an allelic variant or splice variant of the nucleotide sequence set forth in SEQ ID NO: 3, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 4;
(c) a nucleotide sequence of SEQ ID NO: 3, (a), or (b) encoding a polypeptide fragment of at least about 25 amino acid residues, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 4;
(d) a nucleotide sequence encoding a polypeptide that has a substitution and/or deletion of 1 to 251 amino acid residues set forth in any of SEQ ID NOS: 3-4 wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 4;
(e) a nucleotide sequence of SEQ ID NO: 3, or (a)-(d) comprising a fragment of at least about 16 nucleotides;
(f) a nucleotide sequence which hybridizes under moderately or highly stringent conditions to the complement of any of (a)-(e), wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO:4; and
(g) a nucleotide sequence complementary to any of (a)-(e).
The invention further provides for an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO: 4 with at least one conservative amino acid substitution, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 4;
(b) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO: 4 with at least one amino acid insertion, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 4;
(c) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO: 4 with at least one amino acid deletion, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 4;
(d) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO: 4 which has a C- and/or N-terminal truncation, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 4;
(e) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO: 4 with at least one modification selected from the group consisting of amino acid substitutions, amino acid insertions, amino acid deletions, C-terminal truncation, and N-terminal truncation, wherein the polypeptide has an activity of the encoded polypeptide set forth in SEQ ID NO: 4;
(f) a nucleotide sequence of (a)-(e) comprising a fragment of at least about 16 nucleotides;
(g) a nucleotide sequence which hybridizes under moderately or highly stringent conditions to the complement of any of (a)-(f), wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 4; and
(h) a nucleotide sequence complementary to any of (a)-(e).
The invention also provides for an isolated polypeptide comprising the amino acid sequence selected from the group consisting of:
(a) the mature amino acid sequence set forth in SEQ ID NO: 4 comprising a mature amino terminus at residue 1, and optionally further comprising an amino-terminal methionine;
(b) an amino acid sequence for an ortholog of SEQ ID NO: 4, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 4;
(c) an amino acid sequence that is at least about 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 percent identical to the amino acid sequence of SEQ ID NO: 4, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 4 as determined using a computer program selected from the group consisting of GAP, BLASTP, BLASTN, FASTA, BLASTA, BLASTX, BestFit, and the Smith-Waterman algorithm;
(d) a fragment of the amino acid sequence set forth in SEQ ID NO: 4 comprising at least about 25 amino acid residues, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 4;
(e) an amino acid sequence for an allelic variant or splice variant of either the amino acid sequence set forth in SEQ ID NO: 4, or at least one of (a)-(c) wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 4.
The invention further provides for an isolated polypeptide comprising the amino acid sequence selected from the group consisting of:
(a) the amino acid sequence set forth in SEQ ID NO: 4 with at least one conservative amino acid substitution, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 4;
(b) the amino acid sequence set forth in SEQ ID NO: 4 with at least one amino acid insertion, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 4;
(c) the amino acid sequence set forth in SEQ ID NO: 4 with at least one amino acid deletion, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 4;
(d) the amino acid sequence set forth in SEQ ID NO: 4 which has a C- and/or N-terminal truncation, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 4; and
(e) the amino acid sequence set forth in SEQ ID NO: 4, with at least one modification selected from the group consisting of amino acid substitutions, amino acid insertions, amino acid deletions, C-terminal truncation, and N-terminal truncation, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO: 4.
Also provided are fusion polypeptides comprising the polypeptide sequences of (a)-(e) above of the preceding paragraphs.
The present invention also provides for an expression vector comprising the isolated nucleic acid molecules set forth herein, recombinant host cells comprising recombinant nucleic acid molecules set forth herein, and a method of producing a Fhm polypeptide comprising culturing the host cells and optionally isolating the polypeptide so produced.
A transgenic non-human animal comprising a nucleic acid molecule encoding a Fhm polypeptide is also encompassed by the invention. The Fhm nucleic acid molecules are introduced into the animal in a manner that allows expression and increased levels of the Fhm polypeptide, which may include increased circulating levels. The transgenic non-human animal is preferably a mammal.
Also provided are derivatives of the Fhm polypeptides of the present invention.
Additionally provided are selective binding agents such as antibodies and peptides capable of specifically binding the Fhm polypeptides of the invention. Such antibodies and peptides may be agonistic or antagonistic.
Pharmaceutical compositions comprising the nucleotides, polypeptides, or selective binding agents of the present invention and one or more pharmaceutically acceptable formulation agents are also encompassed by the invention. The pharmaceutical compositions are used to provide therapeutically effective amounts of the nucleotides or polypeptides of the present invention. The invention is also directed to methods of using the polypeptides, nucleic acid molecules, and selective binding agents. The invention also provides for devices to administer a Fhm polypeptide encapsulated in a membrane.
The Fhm polypeptides and nucleic acid molecules of the present invention may be used to treat, prevent, ameliorate, diagnose and/or detect diseases and disorders, including those recited herein. Expression analysis in biological, cellular or tissue samples suggests that Fhm polypeptide may play a role in the diagnosis and/or treatment of TNF-related diseases including, but not limited to, acquired-immunodeficiency syndrome (AIDS), anemia, autoimmune diseases, cachexia, cancer, cerebral malaria, diabetes mellitus, disseminated intravascular coagulopathy, erythryoid sick syndrome, hemorrhagic shock, hepatitis, insulin resistance, leprosy, leukemia, lymphoma, meningitis, multiple sclerosis, myocardial ischaemia, obesity, rejection of transplanted organs, rheumatoid arthritis, septic shock syndrome, stroke, adult respiratory distress syndrome (ARDS), tuberculosis, and a number of viral diseases. This expression can de detected with a diagnostic agent such as Fhm nucleotide.
The invention encompasses diagnosing a pathological condition or a susceptibility to a pathological condition in a subject caused by or resulting from abnormal levels of Fhm polypeptide comprising determining the presence or amount of expression of the Fhm polypeptide in a sample; and comparing the level of said polypeptide in a biological, tissue or cellular sample from either normal subjects or the subject at an earlier time, wherein susceptibility to a pathological condition is based on the presence or amount of expression of the polypeptide.
The present invention also provides a method of assaying test molecules to identify a test molecule which binds to a Fhm polypeptide. The method comprises contacting a Fhm polypeptide with a test molecule and to determine the extent of binding of the test molecule to the polypeptide. The method further comprises determining whether such test molecules are agonists or antagonists of a Fhm polypeptide. The present invention further provides a method of testing the impact of molecules on the expression of Fhm polypeptide or on the activity of Fhm polypeptide.
Methods of regulating expression and modulating (i.e., increasing or decreasing) levels of a Fhm polypeptide are also encompassed by the invention. One method comprises administering to an animal a nucleic acid molecule encoding a Fhm polypeptide. In another method, a nucleic acid molecule comprising elements that regulate or modulate the expression of a Fhm polypeptide may be administered. Exampless of these methods include gene therapy, cell therapy, and anti-sense therapy as further described herein.
Surprisingly, a Fhm polypeptide was highly expressed in a wide range of primary human tumors. Therefore, the present polypeptide, and its useful nucleid acid intermediates, have demonstrated utility in differentiating transformed cells from the background.
In another aspect of the present invention, the Fhm polypeptides may be used for identifying receptors or binding partners thereof (xe2x80x9cFhm receptorsxe2x80x9d or xe2x80x9cFhm binding partnersxe2x80x9d). Various forms of xe2x80x9cexpression cloningxe2x80x9d have been extensively used to clone receptors for protein or co-factors. See, for example, Simonsen and Lodish, Trends in Pharmacological Sciences, 15: 437-441, 1994, and Tartaglia et al., Cell, 83:1263-1271, 1995. The isolation of the Fhm receptor(s) or Fhm binding partner(s) is useful for identifying or developing novel agonists and antagonists of the Fhm polypeptide-signaling pathway.
Such agonists and antagonists include soluble Fhm ligand(s), anti-Fhm selective binding agents (such as Fhm antibodies and derivatives thereof), small molecules, peptides or derivatives thereof capable of binding Fhm polypeptides, or antisense oligonucleotides, any of which can be used for potentially treating one or more diseases or disorders, including those recited herein.
In certain embodiments, a Fhm polypeptide agonist or antagonist may be a protein, peptide, carbohydrate, lipid, or small molecular weight molecule which interacts with Fhm polypeptide to regulate its activity.
In another aspect of the present invention, the Fhm polypeptides may be used for identifying receptors thereof (xe2x80x9cFhm receptorsxe2x80x9d). Various forms of xe2x80x9cexpression cloningxe2x80x9d have been extensively used for cloningto clone receptors for protein ligands. See for example, H. Simonsen and H. F. Lodish, Trends in Pharmacological Sciences, vol. 15, 437-44115:437-44, 1994, and Tartaglia et al., Cell, 83:1263-1271, 1995. The isolation of the Fhm receptor(s) is useful for identifying or developing novel agonists and antagonists of the Fhm polypeptide-signaling pathway. Such agonists and antagonists include soluble Fhm receptor(s), anti-Fhm receptor selectivereceptor-selective binding agents (such as antibodies and derivatives thereof), small molecules, and antisense oligonucleotides, any of which can be used for treating one or more of the diseases or disorders, including those recited herein.